1. Field of the Invention
The present invention relates to polypeptides and more specifically to cecropin-like polypeptides with various C-terminus modifications. These modified Cecropin-like polypeptides exhibit increased potency and broader spectrum of antimicrobial activity than the cecropins. This invention also relates to methods of producing these modified cecropins and methods of suppressing the growth of Gram-negative or Gram-positive bacteria by their administration.
2. Brief Description of the Background Art
The immune system of the Cecropia moth and several lepidopteran insects is characterized by an effective humoral response which is mainly associated with the cecropins, a recently discovered family of antibacterial peptides (Boman, H. G. and Steiner, H., Current Topics In Microbiology and Immunology 94/95:75-91 (1981). Three major cecropins, A, B, and D, have been purified from immune hemolymph and their sequences have been elucidated (Qu et al., European Journal of Biochemistry 127:219-224 (1982); Hultmark et al., Ibid, 127:207-217 (1982); and Hultmark et al., U.S. Pat. Nos. 4,355,104 and 4,520,016). All cecropins are small basic peptides with a high degree of mutual sequence homology. The amino acid sequences of cecropins A, B and D from Antheraea pernyi (A.p.) and from Hylophora cecropia (H.c.) are as follows: ##STR1##
The natural cecropins possess antibacterial activity against a variety of bacteria including both Gram-negative and Gram-positive bacteria. The available data on the mode of action of the cecropins indicate that they disrupt the cytoplasmic membranes of bacteria (Steiner et al., Nature 292:246-248 (1981)). It is apparent from the literature that different bacterial species have different sensitivities to the cecropins, and that each cecropin has a distinct spectrum of activity. For example, Bacillus megaterium is highly sensitive to cecropins A and B, but relatively resistant to cecropin D. Both Gram-negative and Gram-positive organisms have been shown to be sensitive to cecropins in the micromolar concentration range. Organisms showing a high level of sensitivity to cecropins include Escherichia coli, Pseudomonas aeruginosa, Bacillus megaterium, and Salmonella typhimurium.
Although cecropins A and B have eight amino acid replacements within the first 32 residues and possess unique C-terminal sequences, their activities against nine different bacterial species are very similar. This suggests that many amino acid substitutions can be tolerated without altering the biological activity of the peptide. Similarly, cecropin B from the Chinese oak silk moth (A. pernyi) differs from cecropin B from North American silk moths (H. cecropia) at four positions; however, three of the changes are replacements for the corresponding amino acids found in the H. cecropia A form. The fourth change is in a position where H. cecropia A and B forms differ and is a conservative change.
The carboxyl terminus in all naturally occurring cecropins is blocked and, in the case of cecropin A, the blocking group R is a primary amide (Andreu et al., P.N.A.S. USA 80:6475-6479 (1983)). Cecropin A and several related peptides have recently been synthesized by solid phase techniques and have been shown to be totally indistinguishable from natural cecropin A by chemical and physical criteria as well as biological activity when the C-terminus was amidated (Andreu et al., supra). We have observed that the modification of the carboxy terminus is required for bactericidal activity in the micromolar concentration range against most Gram-positive and some Gram-negative bacteria.
The covalent modification of the carboxy terminus of proteins can be achieved quantitatively by the cleavage at methionyl residues with cyanogen bromide. This chemical cleavage results in the formation of C-terminal homoserine lactone which can be modified by a variety of chemical agents (Horn, Analytical Biochemistry 69:583-589 (1975); Kempe et al., Gene 39:239-245 (1985); Kempe et at., Biotechnology 4:565-568 (1986).
In view of the great usefulness of the cecropins and analogs thereof and of the great promise that recombinant DNA methods offer for the production of proteins, it appeared desirable to provide a system for the production of cecropin-like polypeptides by means of such technology. Furthermore, it would be desirable to produce recombinant cecropin-like polypeptides that possess bactericidal activity against both Gram-negative and Gram-positive bacteria as do naturally occurring cecropins.